Purines such as adenosine have been shown to play a wide array of roles in biological systems. For example, physiological roles played by adenosine include, inter alia, modulator of vasodilation and hypotension, muscle relaxant, central depressant, inhibitor of platelet aggregation, regulator of energy supply/demand, responder to oxygen availability, neurotransmitter, and neuromodulator. (Bruns, Nucleosides & Nucleotides, 10(5), 931-934 (1991)). Because of its potent actions on many organs and systems, adenosine and its receptors have been the subject of considerable drug-development research (Daly, J. Med. Chem., 25, 197 (1982)). Potential therapeutic applications for agonists include, for instance, the prevention of reperfusion injury after cardiac ischemia or stroke, and treatment of hypertension and epilepsy (Jacobson, et al., J. Med. Chem., 35, 407-422 (1992)). Adenosine itself has recently been approved for the treatment of paroxysmal supra ventricular tachycardia (Pantely, et al., Circulation, 82, 1854 (1990)).
The present invention relates to the neurotransmitter role of purines, in particular, to the purine nucleotide adenosine-5'-triphosphate (ATP), and certain analogs thereof. The neurotransmitter role of ATP is mediated by certain receptors, known collectively as P.sub.2 -purinergic receptors ("P.sub.2 receptors"). P.sub.2 receptors are more responsive to ATP and the diphosphate form of adenosine (ADP) than they are to the monophosphate form of adenosine (AMP) and adenosine itself. Other aspects of these receptors relevant to an appreciation of the present invention include the fact that they are not antagonized by methylxanthines, a compound that is structurally related to ATP. Nor do these receptors act via an adenylate cyclase system, as is the case of other neurotransmitters (see Burnstock, Drug Develop. Res., 28, 195-206 (1993)).
P.sub.2 receptors have been sub-classified into several major nucleotide receptor subtypes on the basis of relative potencies of ATP analogs and on selective antagonism (Burnstock et al., Gen. Pharmacol., 16, 433-440 (1985a); Gordon, Biochem. J., 233, 309-319 (1986)). For example, P.sub.2x receptors are activated by .alpha.,.beta.-methylene-ATP and apparently consist of ligand-gated cation channels (Benham et al., Nature, 328, 275-278 (1987); Benham, J. Physiol., 419, 689-701 (1989); and Bean, Trends Pharmacol. Sci., 13, 87-90 (1992)). P.sub.2Y -receptors are activated by 2-methylthio-ATP and are linked to second messengers via G-proteins. The principal second messenger system activated by P.sub.2Y receptors is the metabolism of phosphatidyl inositol (Harden et al., Biochem. J., 252, 583-593 (1988); Boyer et al., J. Biol. Chem., 264, 884-890 (1989); Pirroton et al., J. Biol. Chem, 262, 17461 (1987); Haggblad et al., Neurosci. Lett., 74, 199-204 (1987)), leading to liberation of intracellular calcium stores, activation of protein kinase C, and increases in cytoplasmic calcium. A less clearly defined subtype of the P.sub.2 receptor family, the P.sub.2U receptor (Dubyak, Amer. J. Respir. Cell. Molec. Biol., 4, 295-300 (1991)), also promotes inositol lipid hydrolysis and is activated by ATP and UTP, but not by many analogs of ATP, UTP, and ADP. Other cell-specific P.sub.2 receptors include P.sub.2T receptors, which regulate platelet cell function (Hoyle et al., in Adenosine in the Nervous System (T. W. Stone, ed., Academic Press, London, 1991), pp. 43-76; Gordon, supra; Hoyle, in Autonomic Neuroeffector Mechanisms (G. Burnstock and C. H. V. Hoyle, eds., Harwood Academic Publishers, 1992), pp. 367-407), and P.sub.2Z receptors, which regulate ion permeability in mast cells, fibroblasts and leukocytes (Hoyle et al., supra; Gordon, supra; Hoyle, supra; Tatham et al., Eur. J. Pharmacol., 147, 13-21 (1988)).
The P.sub.2X and P.sub.2Y receptors are widely distributed subtypes, being found on smooth muscle cells of the cardiovascular, gastrointestinal and genitourinary systems, and cardiac muscle, and many diverse cell types, including: endothelial cells, hepatocytes, erythrocytes, pancreatocytes, pulmonary alveolar cells, autonomic ganglionic neurons, sensory neurons, and also within the central nervous system (see Hoyle et al., supra; Hoyle, supra).
The P.sub.2X receptor mainly mediates constriction of smooth muscle in visceral organs, such as urinary bladder, vas deferens, and blood vessels (Burnstock, Nucleos. & Nucleot., 10, 917-930 (1991)). The endogenous ligand for this receptor is probably ATP, which was demonstrated in the work of Suet al. (Science, 173, 336-338 (1971)). Several ATP derivatives, such as the aforementioned .alpha.,.beta.-methylene ATP, have been demonstrated to have greater selectivity and potency than ATP itself, all of which have a modified triphosphate chain (see Bo et al., Gen. Pharmac., 24, 637-640 (1993)). The results of various earlier studies, commented upon by Bo et al., supra, indicate that modifications of the triphosphate chain improve the efficacy of certain agonists of the P.sub.2X receptor. The results of Bo et al. confirmed this observation and extended it by indicating that the polyphosphate chain was also responsible for the affinity of ATP for the P.sub.2X receptor.
The P.sub.2Y receptor appears to have opposite effects relative to the P.sub.2X receptor in that activation of the P.sub.2Y receptor with extracellular ATP induces relaxation of visceral and vascular smooth muscle. In particular, the response of P.sub.2Y activation has been studied in the taenia coli, the aorta, erythrocyte membranes, and other systems of study. Interactions of certain ligands with P.sub.2Y receptors often is correlated with phospholipase C activity, which is discussed further hereinbelow.
Pharmacological, biochemical, and structural characterization of P.sub.2 receptors and their natural and synthetic ligands has been relatively limited, due to a multiplicity of biological effects and inconsistencies in the potency of "selective" agents (see Inoue et al.,News in Physiol. Sci., 2, 56-58 (1992); Silinsky, The Neurosciences, 1, 155-165 (1989)), and the great difficulties in synthesis and purification of nucleoside triphosphates and their analogs. In view of the early results that indicates that only diphosphorylated and triphosphorylated species have activity at the P.sub.2 receptors, the identification of truly selective agents for one of the P.sub.2 receptors may not result in readily applicable pharmaceuticals because of the difficulty in keeping such molecules from rapidly being broken down in the ordinary course of ATP metabolism. Regarding the pursuit of candidate antagonist compounds, one research report states that "[i]n spite of a considerable effort to produce new ATP derivatives and, in particular, to produce antagonists to P.sub.2 R [i.e., P.sub.2 receptors], not one selective P.sub.2 R subtype antagonist has so far been obtained" (Zimmet et al., Nucleos. & Nucleot., 12(1), 1-20 (1993)). Accordingly, drug development that would capitalize on the P.sub.2 receptor biology has been slow in realization.
In view of the background recited hereinabove, it is an object of the present invention to provide new P.sub.2 receptor ligands. Such ligands preferably would have the advantages of being selective for a single subclass, being relatively easy to synthesize, having a charge suitable for crossing cell membranes readily, being resistant to cleavage consequent to ATP metabolism, and having activity that is greater than that of ATP itself. It is a further object of the present invention to provide pharmaceutical compositions that incorporate the newly disclosed receptor ligands. Yet a further object of the present invention is to provide methods of use of the disclosed receptor ligands in the in vivo treatment of various disease conditions that can be treated by manipulation of P.sub.2 receptor function.
These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.